The invention discloses a genomic analysis process, in particular for analysing and localising hereditary properties in the genome. This process has applications in a large number of fields, in particular in medicine, agriculture, forensic medicine and fundamental research.
1.1 Definition of Microsatellites
Here, the term of a microsatellite is used in the following sense: a microsatellite is an oligonucleotide repeat showing various alleles between the individual chromosomes of a species, i.e. is polymorphic. The various alleles differ by their length, i.e. their molecular weight. Each microsatellite is flankedxe2x80x94and thus also definedxe2x80x94by two non-polymorphic DNA sequences appearing only once which as hybridisation partners are used by the primers for carrying out a polymerase chain reaction.
1.2 Applications of Microsatellite Markers
Microsatellite markers are a main instrument of modern genetic analysis.
1.2.1
On the one hand, these markers are applied to detect after a meiotic cell division took place which of the two alleles was transferred to a descendant in a genomic locus associated with the respective marker.
This principle is used in the coupling analysis: If microsatellite marker alleles, together with a genetic defect, are inherited with a frequency above average in a family with several persons suffering from a hereditary disease the defect locus and the microsatellite marker are physically coupled, i.e. they are situated close to each other in the genome.
With localising of a microsatellite in a genome being known this information allows to draw conclusions as to the position of the gene(s) responsible for the defect. For the time being, two methods of the coupling analysis are applied in practise:
the so-called lod-score method based on an analysis of a few up to numerous large families involving a few persons affected,
the xe2x80x9caffected relative methodxe2x80x9d (ARM) based on a comparison of a multitude of pairs of related test persons.
In both cases, haplotyping of numerous persons is required. By haplotyping we understand detecting for each person to be analysed which of the alleles of each of the polymorphic marker is contained in the genome of the test person. Today, nearly exclusively microsatellite markers are typified. This is due to the fact that they are analysed most reliably and rapidly (see semiautomatization below) and may be identified most efficiently. That is why according to the state of the art more than 5000 of these markers are available for man and all systems available on the market for carrying out the coupling analysis are based on these markers. To establish with some degree of certainty a coupling of a microsatellite to form genes which are not responsible for a defect at least 300-600 pairs of affected relatives will have to be examined with always at least 400 microsatellites in the case of a disease not inherited according to Mendel""s pattern (This group of defects and predispositions is the main target of our method).
Genotypifying of by far more test persons with essentially more markers would be ideal to get a possibly exact idea of the genomic loci after establishing the coupling groups. In this second step markers covering the regions detected in the first step by coupling with an essentially higher resolution are applied.
The most up-to-date and fastest approach to genotypification with microsatellites is using florescence-marked microsatellite primers and determining the size of microsatellite alleles by means of a semiautomatic sequencer. This method allows to analyse up to 24, yet on an average, only 15 microsatellites on an electrophoresis gel path (with up to 48 patris per gel) which, however, means that in the best case 1000-2000 of such gels are used to establish a coupling applying the ARM (Vignal, A. et al., 1993; Methods in Molecular Genetics: Gene and Chromosome analysis. Academic Press, San Diego. Pages 211-221 and Davies, J. L. et al., 1994; Nature 371: 130-136). This means, that a sequencer would be utilised to the full for at least one year in the ideal case. The costs of such a coupling analysis including personnel costs may total up to a few millions of DM. In the last few years the interest of the participating scientists was focussed ever more on frequently occurring, polygenic, multifactorial or multigene diseases (Lathrop, G. M., 1993; Current Opinion in Biotechnology 4:678-683). The more components will participate in the development of a disease the more persons have to be genotypified to establish a coupling. Thus, the expenses and costs will go up exponentially with the complexity of a disease. That means, that a coupling analysis for the so-called widespread diseases will be by far more expensive than has been mentioned before.
In the case of useful and domestic animals, yet also plants, the coupling analysis is the most promising instrument to localise valuable breeding properties in the genome and to isolate the respective genes.
This allows the breeder to concentrate positive properties and to minimise negative properties. The breeding strategy common so far allows to assess a breeding success only after a few generations have grown up. In addition, the breeding result is not foreseeable in most of the cases.
Notably, in the case of positive properties such as stable resistances to pathogenes and resistance to cold, as a rule, comprehensive genetic parameters are concerned requiring an expenditure on the human genetic problems as described above.
1.2.2
Secondly, there may be detected whether a certain allele of a microsatellite is contained at all in two genomes to be compared which after having analysed sufficient markers allows to confirm or to exclude a relation. As to man, this question arises e.g. in the case of paternity tests or in forensic medicine to exclude or identify offenders. In addition to typifying HLA surface antigenes here, the use of highly polymorphic microsatellite markers gained acceptance in practice and replaced largely genetic dactylograms (multiloci).
Also in the case of useful animals the proof of origin by means of a microsatellite analysis was declared the standard method, on international scale.
For these applications, as a rule, about 10 microsatellite markers are analysed. The whole proof including evaluation will require about 2 days.
For the time being, the costs of such an analysis total at least DM 50, also in the case of the quantity of samples being large.
2.1 Coupling Analysis of Complex Diseases of Man
An analysis of up to a few thousands of pairs of related persons affected is required for analysing widespread, multifactorial diseases such as e.g. arteriosclerosis, diabetes II, Alzheimer or schizophrenia as it is, as a rule, not possible to apply the lod-score method. The main reasons for that are a lack of large families involving sufficient affected persons and a lack of a hereditary transmission model process. That is why only extremely few research groups are worldwide in a position to carry out such an analysis. The expenses of such an analysis are very high, as described above in greater detail. Yet, the equipment is also expensive because the production costs of fluorescence-marked primers are extremely high. To synthesize these primers we depend on a few companies.
A principle disadvantage of the electrophoretic separation of fluorescence-marked DNA fragments consists in the fact that a proper automatic sample analysing is not possible. Automating the evaluation is still more difficult. The software designed by the producers of sequencers is necessarily insufficient. Basically, efforts are made to automate a method which is not to be automated with the aid of most complicated computers.
A decisive disadvantage of the common electrophoretic methods is that an absolute determination of the size of fragments is not possible.
This means, that expensive and finally not always reliable calibration procedures are required. Attempts are made to eliminate these problems partly by using cloned alleles as internal standards. This might be achieved also limitedly.
Yet, the circumstance that the mobility of the DNA fragments corresponding to the alleles depends on various parameters of sample analysing, thus e.g. marking and individual conditions of reaction in the PCR. Thus, the decisive measurable variable of a microsatellite analysis, namely the length of the fragments, has to be based on a number of auxiliary variables. Yet, it is only possible to assign a certain frequency to a reliably identified allele in an analyses of identity or origin. However, the reliability of the data gained in an analysis depends nearly exclusively on the last-mentioned.
2.2 Coupling Analysis in the Case of Useful Animals and Plants
As a rule, localising of genes of useful and domestic animals and plants which control valuable breeding properties is impossible owing to the extremely high price of the study. The vast majority of the interesting breeding properties has a polygenic background. Thus, a very expensive coupling analysis would be required.
2.3 Proof of Origin of Useful Animals
Also here, the decisive factor is the price of an analysis. A proof of origin of useful animals will be appropriate if each animal will be individually genotypified or at least analysing of a large quantity of random samples will be possible. The price of a conventional microsatellite analysis has an essential share in the price of an animal for most of the species of useful animals.
Neither breeders nor consumers will be prepared to accept an essential increase in the price of useful animals which would make a microsatellite analysis of these animals senseless from a market economy perspective.
The invention is based on the task to rationalise the genomic analysis, in particular localising hereditary properties in the genome, thus allowing its application also in cases where previous methods are not taken into consideration for reasons of costs.
The process according to the invention is characterised by the principle claim, the subclaims are preferential variants. As an essential component it envisages analysing of microsatellites by means of mass spectrometry. In this connection, the unique sequences flanking each of the microsatellites as a single-line DNA shall be immobilised on a solid matrix. The matrix is subdivided in a way that each single-line DNA defining a microsatellite is immobilised in a certain position of this matrix. If a mixture of amplified microsatellites in a single-line state is placed on this matrix the available alleles of each of the microsatellites hybridise only exactly in the position of the matrix where the primer sequence defining the microsatellite had been marked.